The invention relates to a novel lipid kinase which is part of the PI3 Kinase (P13K) family and more specifically the invention relates to various aspects of the novel lipid kinase particularly, but not exclusively, to an identification of expression of said kinase with a view to diagnosing or predicting motility or invasion of cells such as metastasis of cancer cells; and also agents for interfering with said expression or inhibiting said kinase with a view to enhancing or reducing or preventing said motility or invasion so as to enhance or restrict, respectively the movement of selected cells.
An overview of the PI3 kinase family of enzymes is given in our co-pending Patent Application WO93/21328. Briefly, this class of enzymes shows phosphoinositide (hereinafter referred to after as PI) 3-kinase activity. Following major advances in our knowledge of cell signal transduction and cell second messenger systems it is known that the PI3Ks have a major role to play in regulating cell function. Indeed, it is known that PI3Ks are members of a growing number of potential signalling proteins which associate with protein-tyrosine kinases activated either by ligand stimulation or as a consequence of cell transformation. Once thus associated they provide an important complex in the cell signalling pathway and thus direct events towards a given conclusion.
PI3 kinases catalyse the addition of phosphate to the 3xe2x80x2-OH position of the inositol ring of inositol lipids generating phosphatidyl inositol monophosphate, phosphatidyl inositol diphosphate and phosphatidyl inositol triphosphate (Whitman et al, 1988, Stephens et al 1989 and 1991). A family of PI3 kinase enzymes has now been identified in organisms as diverse as plants, slime molds, yeast, fruit flies and mammals (Zvelebil et al, 1996).
It is conceivable that different PI3 kinases are responsible for the generation of the different 3xe2x80x2-phosphorylated inositol lipids in vivo. Three classes of PI3 kinase can be discriminated on the basis of their in vitro lipid substrates specificity. Enzymes of a first class have a broad substrate specificity and phosphorylate PtdIns, PtdIns(4)P and PtdIns(4,5)P2. Class I PI3 kinases include mammalian p110xcex1, p110xcex2 and p110xcex3 (Hiles et al, 1192; Hu et al, 1993; Stephens et al, 1994; Stoyanov et al, 1995).
P110xcex1 and p110xcex2 are closely related PI3 kinases which interact with p85 adaptor proteins and with GTP-bound Ras.
Two 85 kDa subunits, p85xcex1 and p85xcex2, have been cloned (Otsu et al, 1992). These molecules contain an N-terminal src homology-3 (SH3) domain, a breakpoint cluster (bcr) homology region flanked by two proline-rich regions and two src homology-2 (SH2) domains. Shortened p85 proteins, generated by alternative splicing from the p85xcex1 gene or encoded by genes different from those of p85xcex1/xcex2, all lack the SH3 domain and the bcr region, which seem to be replaced by a unique short N-terminus (Pons et al, 1995; Inukai et al, 1996; Antonetti et al, 1996). The SH2 domains, present in all p85 molecules, provide the heterodimeric p85/p110 PI3Ks with the capacity to interact with phosphorylated tyrosine residues on a variety of receptors and other cellular proteins. In contrast to p110xcex1 and xcex2, p110xcex3 does not interact with p85 but instead associates with a p101 adaptor protein (Stephens et al, 1996). P110xcex3 activity is stimulated by G-protein subunits.
PI3Ks of a second class contains enzymes which, at least in vitro, phosphorylate PtdIns and PtdIns(4)P but not PtdIns(4, 5)P2 (MacDougall et al, 1995; Virbasius et al, 1996, Molz et al, 1996). These PI3Ks all contain a C2 domain at their C-terminus. The in vivo role of these class II PI3Ks is unknown.
A third class of PI3K has a substrate specificity restricted to PtdIns. These PI3Ks are homologous to yeast Vps34p which is involved in trafficking of newly formed proteins from the Golgi apparatus to the vacuole in yeast, the equivalent of the mammalian lysosome (Stack et al, 1995). Both yeast and mammalian Vps34p occur in a complex with Vps15p, a 150 kDa protein serine/threonine kinase (Stack et al, 1995; Volinia et al, 1995; Panaretou et al, submitted for publication).
PtdIns(3)P is constitutively present in cells and its levels are largely unaltered upon extracellular stimulation. In contrast, PtdIns(3, 4)P2 and PtdIns(3, 4, 5)P3 are almost absent in quiescent cells but are produced rapidly upon stimulation by a variety of growth factors, suggesting a likely function as second messengers (Stephens et al, 1993). The role of PI3Ks and their phosphorylated lipids in cellular physiology is just beginning to be understood. These lipids may fulfill a dual role: apart from exerting physical, charge-mediated effects on the curvature of the lipid bilayer, they also have the capacity to interact with specific binding proteins and modulate their localisation and/or activity. Amongst the potential targets for these lipids are protein kinases such as protein kinase C isoforms, protein kinase N/Rho-activated kinases and Akt/RAC/protein kinase B (Toker et al, 1994; Palmer et al, 1995; Burgering and Coffer, 1995; Franke et al, 1995; James et al, 1996; Klippel et al, 1996). Akt/RAC/protein kinase B is likely to be upstream of targets such as p70 S6 kinase and glycogen synthase kinase-3 (Chung et al, 1994; Cross et al, 1995). PI3Ks also affect the activity of small GTP-binding proteins such as Rac and Rab5, possibly by regulating nucleotide exchange (Hawkins et al, 1995; Li et al, 1996). Ultimately, the combination of these actions can result in cytoskeletal rearrangements, DNA synthesis/mitogenesis, cell survival and differentiation (Vanhaesebroeck et al, 1996).
We describe herein a mammalian novel Class I PI3 Kinase which we have termed p110xcex4. This novel PI3 Kinase typifies the Class I PI3 Kinase family in that it binds p85xcex1, p85xcex2 and p85xcex3. In addition, it also binds GTP-ras but, like p110xcex1, shows no binding of rho and rac. It also shares the same GTP-broad phosphoinositide lipid substrate specificity of p110xcex1 and p110xcex2, and it also shows protein kinase activity and has a similar drug sensitivity to p110xcex1.
However, it is characterised by its selective tissue distribution. In contrast to p100xcex1 and p110xcex2 which seem to be ubiquitously expressed, p110xcex4 expression is particularly high in white blood cell populations i.e. spleen, thymus and especially peripheral blood leucocytes. In addition to this observation we have also found that p110xcex4 is expressed in most melanomas, but not in any melanocytes, the normal cell counterpart of melanomas. Given the natural distribution of p110xcex4 in tissues which are known to exhibit motility or invasion and also the expression of p110xcex4 in cancer cells we consider that p110xcex4 has a role to play in cell motility or invasion and thus the expression of this lipid kinase in cancer cells can explain the metastatic behaviour of cancer cells.
A further novel feature of p110xcex4 is its ability to autophosphorylate in a MN2+-dependent manner. Indeed, we have shown that autophosphorylation tends to hinder the lipid kinase activity of the protein. In addition, p110xcex4 contains distinct potential protein:protein interaction modules including a proline-rich region (see FIG. 1, position 292-311, wherein 8 out of 20 amino acids are proline) and a basic region leucine zipper (bZIP) like domain (Ing et al., 1994 and Hirai et al., 1996). Such biochemical and structural differences between p85-binding PI3 kinases indicate that they may fulfill distinct functional roles and/or be differentially regulated in vivo.
We disclose herein a nucleic acid molecule, of human origin, and corresponding amino acid sequence data relating to p110xcex4. Using this information it is possible to determine the expression of p110xcex4 in various tissue types and in particular to determine the expression of same in cancer tissue with a view to diagnosing the motility or invasiveness of such tissue and thus predicting the potential for secondary tumours occurring. Moreover, it will also be possible to provide agents which impair the expression of p110xcex4 or alternatively interfere with the functioning of same. For example, having regard to the sequence data provided herein it is possible to provide antisense material which prevents the expression of p110xcex4.
As mentioned above, the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a PI3Kxcex4 protein, to decrease transcription and/or translation of PI3Kxcex4 genes. This is desirable in virtually any medical condition wherein a reduction in PI3Kxcex4 gene product expression is desirable, including to reduce any aspect of a tumor cell phenotype attributable to PI3Kxcex4 gene expression. Antisense molecules, in this manner, can be used to slow down or arrest such aspects of a tumor cell phenotype.
As used herein, the term xe2x80x9cantisense oligonucleotidexe2x80x9d or xe2x80x9cantisensexe2x80x9d describes an oligoneucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon the DNA sequence presented in FIG. 9 or upon allelic or homologous genomic and/or DNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and more preferably, at least 15 consecutive bases which are complementary to the target. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5xe2x80x2 upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3xe2x80x2-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457. 1994) and at which proteins are not expected to bind. Finally, although FIG. 9 discloses cDNA sequence, one of ordinary skill in the art may easily derive the genomic DNA corresponding to the cDNA of FIG. 9. Thus, the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to FIG. 9. Similarly, antisense to allelic or homologous DNAs and genomic DNAs are enabled without undue experimentation.
In one set of embodiments, the antisense oligonucleotides of the invention may be composed of xe2x80x9cnaturalxe2x80x9d deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5xe2x80x2 end of one native nucleotide and the 3xe2x80x2 end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
In preferred embodiments, however, the antisense oligonucleotides of the invention also may include xe2x80x9cmodifiedxe2x80x9d oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
The term xe2x80x9cmodified oligonucleotidexe2x80x9d as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5xe2x80x2 end of one nucleotide and the 3xe2x80x2 end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.
The term xe2x80x9cmodified oligonucleotidexe2x80x9d also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3xe2x80x2 position and other than a phosphate group at the 5xe2x80x2 position. Thus modified oligonucleotides may include a 2xe2x80x2-0-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. Modified oligonucleotides also can include base analogs such as C-5 propyne modified bases (Wagner et al., Nature Biotechnology 14:840-844, 1996). The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding PI3Kxcex4 proteins, together with pharmaceutically acceptable carriers.
Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term xe2x80x9cpharmaceutically acceptablexe2x80x9d means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term xe2x80x9cphysiologically acceptablexe2x80x9d refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
It is therefore an object of the invention to identify a novel PI3 Kinase and so provide means for predicting the likely motility or invasiveness of cells.
It is a yet further object of the invention to provide agents that enhance or reduce or prevent the expression of p110xcex4 and/or agents which interfere with the functioning of p110xcex4, with a view to enhancing or hindering or preventing, respectively, the motility or invasiveness of cells.
According to a first aspect of the invention there is therefore provided an isolated autophosphorylating polypeptide which possesses PI3 kinase activity.
Ideally said polypeptide is derived from white blood cells and is typically expressed in melanomas, more ideally still said polypeptide is of human origin.
Moreover, the polypeptide is capable of association with p85 subunits of mammalian PI3 Kinases ideally to produce active complexes.
More preferably still the polypeptide has the amino acid sequence shown in FIG. 1A or a sequence homologous thereto which is in particularly characterised by a proline rich domain.
Reference herein to the term homologous is intended to cover material of a similar nature or of common descent or pocessing those features, as herein described, that characterise the protein, or material, whose corresponding nucleic acid molecule hybridises, such as under stringent conditions, to the nucleic acid molecule shown in FIG. 9. Typical hybridisation conditions would include 50% formamide, 5xc3x97SSPE, 5xc3x97Denhardts solution, 0.2% SDS, 200 xcexcg/ml denatured sonicated herring sperm DNA and 200 xcexcg/ml yeast RNA at a temperature of 60xc2x0 C., (conditions described in the published patent specification WO 93/21328).
Ideally the polypeptide is produced using recombinant technology and is typically of human origin.
According to a further aspect of the invention there is provided an antibody to at least a part of the polypeptide of the invention, which antibody may be polyclonal or monoclonal.
According to a further aspect of the invention there is provided the whole or a part of the nucleic acid molecule shown in FIG. 9 which molecule encodes an autophosphorylating polypeptide having PI3 Kinase activity.
In the instance where said part of said molecule is provided, the part will be selected having regard to its purpose, for example it may be desirable to select a part having kinase activity for subsequent use or another part which is most suitable for antibody production.
According to a further aspect of the invention there is provided a nucleic acid molecule construct comprising a whole or a part of the nucleic acid molecule of the invention wherein the latter nucleic acid molecule is under the control of a control sequence and in appropriate reading frame so as to ensure expression of the corresponding protein.
According to a yet further aspect of the invention there is provided host cells which have been transformed, ideally using the construct of the invention, so as to include a whole or a part of the nucleic acid molecule shown in FIG. 9 so as to permit expression of a whole, or a significant part, of the corresponding polypeptide.
Ideally these host cells are eukaryotic cells for example, insect cells such as cells from the species Spodoptera frugiperda using the baculovirus expression system. This expression system is favoured in the instance where post translation modification is required. If such modification is not required a prokaryotic system may be used.
According to a further aspect of the invention there is provided a method for diagnosing the motility of cells comprising examining a sample of said cells for the expression of the polypeptide of the invention.
Ideally, investigations are undertaken in order to establish whether mRNA corresponding to the polypeptide of the invention is expressed in said cells, for e.g. by using PCR techniques or Northern Blot analysis. Alternatively, any other conventional technique may be undertaken in order to identify said expression.
According to a yet further aspect of the invention there is provided a method for identifying antagonists effective at blocking the activity of the polypeptide of the invention which comprises screening candidate molecules for such activity using the polypeptide, or fragments thereof the invention.
Ideally, screening may involve artificial techniques such as computer-aided techniques or conventional laboratory techniques.
Ideally, the above method is undertaken by exposing cells known to express the polypeptide of the invention, either naturally or by virtue of transfection, to the appropriate antagonist and then monitoring the motility of same.
Alternatively, the method of the invention may involve competitive binding assays in order to identify agents that selectively and ideally irreversibly bind to the polypeptide of the invention.
According to a yet further aspect of the invention there is provided a pharmaceutical or veterinary composition comprising an agent effective at enhancing or blocking the activity or expression of the polypeptide of the invention which has been formulated for pharmaceutical or veterinary use and which optionally also includes a dilutant, carrier or excipient and/or is in unit dosage form.
According to a yet further aspect of the invention there is provided a method for controlling the motility of cells comprising exposing a population of said cells to either an agonist or antagonist or the polypeptide of the invention or to antisense material as hereindescribed.
Alternatively, in the aforementioned method said cells may be exposed alternatively or additionally, to the polypeptide of the invention with a view to increasing the effective levels of said polypeptide and so enhancing cell motility.
The aforementioned method may be undertaken either in vivo or in vitro.
According to a yet further aspect of the invention there is provided use of an agent effective at blocking the activity of the polypeptide of the invention for controlling cell motility.
According to a yet further aspect of the invention there is provided use of the polypeptide of the invention for enhancing cell motility.
According to a yet further aspect of the invention there is provided antisense oligonucleotides ideally modified as hereindescribed, for hybridizing to the nucleic acid of the invention.